It was recently discovered that arisugacin, a natural product isolated from a culture of Penicillium, is an inhibitor of acetylcholinesterase (AChE), and on this basis arisugacin has been predicted to be effective in the treatment of Alzheimer""s disease. Related compounds also showed inhibitory activity. Omura, S., et al. (1995), xe2x80x9cArisugacin, a Novel and Selective Inhibitor of Acetylcholinesterase from Penicillium sp. FO-4259,xe2x80x9d J. Antibiotics 48:745-746. Arisugacin and the related compounds are tetracyclic pyrones (having four fused rings). Other tetracyclic pyrones, certain pyripyropenes, have been shown to be inhibitors of cholesterol acyltransferase (ACAT), and therefore have been predicted to be effective in the treatment of atherosclerosis and hypercholesterolemia. Omura, S., et al. (1993), xe2x80x9cPyripyropenes, Highly Potent Inhibitors of Acyl-CoA; Cholesterol Acyltransferase Produced by Aspergillus fumigatus,xe2x80x9d J. Antibiotics 46:1168-1169; and xe2x80x9cKim, Y. K. et al. (1994), xe2x80x9cPyripyropenes, Novel Inhibitors of Acyl-CoA:Cholesterol Acyltransferase Produced by Aspergillus fumigatus,xe2x80x9d J. Antibiotics 47:154-162. Pyripyropene A, one such inhibitor, is further characterized in Tomoda, H., et al. (1994), xe2x80x9cRelative and Absolute Stereochemistry of Pyripyropene A, A Potent, Bioavailable Inhibitor of Acyl-CoA:Cholesterol Acyltransferase (ACAT),xe2x80x9d J. Am. Chem. Soc. 116:12097-12098.
A number of multicyclic pyrones are known to the art and described in Chemical Abstracts; however, tricyclic and tetracyclic pyrones as disclosed and claimed herein, appear not to have been previously described.
There is a need for simpler inhibitors of AchE and ACAT that are useful as treatments for Alzheimer""s disease, atherosclerosis and hypercholesterolemia.
The tricyclic and tetracyclic pyrones of this invention are useful as inhibitors of AChE and ACAT, and can be used in the treatment of Alzheimer""s disease, atherosclerosis and Kit hypercholesterolemia. The tricyclic compounds are also potent inhibitors of cancer cell growth and macromolecule synthesis (e.g., DNA, RNA and protein synthesis) and can be used in the treatment of various forms of cancers including leukemia, ascites, and solid tumors. Further, their short-term inhibition of macromolecule synthesis is reversible following removal, but their long-term inhibition of tumor cell growth is not. Importantly, the tricyclic compounds are also powerful inhibitors of tubulin polymerization and may be useful as cell cycle-specific anticancer drugs. As hereinafter described, certain of these pyrones are useful intermediates in the synthesis of other pyrones of this invention. The tricyclic compounds are cytostatic but not overly cytotoxic.
The tricyclic pyrones of this invention include compounds selected from the group of compounds of the formula: 
wherein:
T is independently CH, N, S or O;
X is independently O, NH or S;
Y is independently O, NH or S;
Z is independently CH, N, S or O;
R1 is independently Formula I; or
R1 and R3and R4 and R5 are, independently, H, OH, alkyl, alkenyl, alkynyl, an aromatic ring system, 
xe2x80x83wherein R and M are independently H, alkyl, alkenyl or alkynyl, an aromatic ring system, amino, amido, sulfhydryl, or sulfonyl, W is Cl, F, Br or OCI, and A is an aromatic ring system;
R2 and R9 are independently H or R where R is as defined above.
As used herein, the term xe2x80x9caromatic ring systemxe2x80x9d includes five and six-membered rings, fused rings, heterocyclic rings having oxygen, sulfur or nitrogen as a ring member, OR-substituted and R-substituted aromatic rings where R is defined as above. Preferably the substituents have one to five carbons. As used herein, the terms xe2x80x9calkyl,xe2x80x9d xe2x80x9calkenyl,xe2x80x9d an xe2x80x9calkynylxe2x80x9d include C1-C6 straight or branched chains. Unless otherwise specified, a general formula includes all stereoisomers.
Compounds of this invention also include compounds of the formula: 
wherein:
X, Y and R2-R3 are as set forth for Formula I;
R1 is independently Formula II or as set forth for Formula I;
R15 is independently NH2, OH, or OCORxe2x80x2 where Rxe2x80x2 is H, or alkyl;
R16 is independently OH or H; and
R15 and R16 taken together are O;
compounds of the formula: 
wherein:
X, Y, T, Z and R2 and R3 are as set forth in Formula I:
R1 is independently Formula III or as set forth for Formula I; and
R6 is H when R7 is OH, or R6 is OH when R7 is H, or R6 and R7 taken together are xe2x95x90O;
compounds of the formula: 
wherein R1 is independently Formula IV or as set forth for Formula I, and R3 is as set forth for Formula I above; and R2, R4 R3 for Formula I above;
and compounds of the formula: 
wherein R1 is Formula V or independently is as set forth for Formula I above.
The tetracyclic pyrones of this invention include compounds selected from the group of compounds of the formula: 
wherein:
R1 and R2 are independently as defined as R3 as set forth for Formula I above;
R10 and R11 and R13 and R14 are independently defined as R3 as set forth for Formula I above; and
R12 is H, alkyl, alkenyl or alkynyl, an aromatic ring system, amino, amido, sulfhydryl, or sulfonyl.
A preferable class of compounds of this invention useful as macromolecule synthesis inhibitors in cancer cells are compounds selected from compounds of the formula: 
wherein:
R1 is independently selected from the group consisting of H, R, 3-pyridyl, R-substituted 3-pyridyl, phenyl, R-substituted, di-substituted and tri-substituted phenyl, Oxe2x80x94R-substituted, di-substituted and tri-substituted phenyl where R is as defined above; and preferably comprises an aromatic ring;
R2 and R9 are independently selected from the group consisting of H and R, where R is as defined above;
R3, R4 and R5 are independently selected from the group H, R, OH, OCHO, and OR where R is as defined above; and
T and Z are independently selected from the group consisting of CH, N, S or O.
Most preferably, the compounds are selected from the group consisting of compounds of Formula 1 wherein:
R1 is independently selected from the group consisting of alkyl, 3-pyridyl and 3,4-dimethoxyphenyl; preferably 3-pyridyl or 3,4-dimethoxyphenyl;
R2 is independently selected from the group consisting of H and CH3;
R3 is independently selected from the group of H, OH, and OCHO;
R4 and R5 are independently H;
R9 is independently selected from the group of H and isopropenyl; and
T and Z are independently CH.
Throughout the specification hereof, chemical structures are depicted and numerically labelled. The names of the numbered structures are set forth in Table 1 and indicated in boldface in the text.
Preferred compounds of this invention are shown in below in Scheme 1 and include compounds selected from the group consisting of: 3-(3-pyridyl)-1H-5a,6,7,8,9-pentahydro-1-oxopyrano[4,3-b]benzopyran [2A]; 3-(3,4-dimethoxyphenyl)-1H-5a,6,7,8,9-pentahydro-1-oxopyrano[4,3-b]benzopyran [3A]; cis- and trans-3-(3,4-dimethoxyphenyl)-5a-methyl-6-formyloxy-1H-5a,6,7,8,9-pentahydro-1-oxopyrano[4,3-b]benzopyran [3D and 3E]; cis- and trans-3-(3-pyridyl)-5a-methyl-6-formyloxy-1H-5a,6,7,8,9-pentahydro-1-oxopyrano[4,3-b]benzopyran [2D and 2E]; cis- and trans-3-(3,4-dimethoxyphenyl)-5a-methyl-6-hydroxy-1H-5a,6,7,8,9-pentahydro-1-oxopyrano[4,3-b]benzopyran [3B and 3C]; 3-methyl-1H-5a,6,7,8,9-pentahydro-1-oxopyrano[4,3-b]benzopyran [1A]; cis- and trans-3-5a-dimethyl-6-formyloxy-1H-5a,6,7,8,9-pentahydro-1-oxopyrano[4,3-b]benzopyran [1D and 1E]; and cis- and trans-3-(3-pyridyl)-5a-methyl-6-hydroxy-1H-5a,6,7,8,9-pentahydro-1-oxopyrano[4,3-b]benzopyran [2B and 2C].
A more preferred class of compounds of this invention includes compounds selected from the group consisting of 3-(3-pyridyl)-1H-5a,6,7,8,9-pentahydro-1-oxopyrano[4,3-b]benzopyran [2A]; 3-(3,4-dimethoxyphenyl)-1H-5a,6,7,8,9-pentahydro-1-oxopyrano[4,3-b]benzopyran [3A]; cis- and trans-3-(3,4-dimethoxyphenyl)-5a-methyl-6-formyloxy-1H-5a,6,7,8,9-pentahydro-1-oxopyrano[4,3-b]benzopyran [3D and 3E]; cis- and trans-3-(3-pyridyl)-5a-methyl-6-formyloxy-1H-5a,6,7,8,9-pentahydro-1-oxopyrano[4,3-b]benzopyran [2D and 2E]; cis- and trans-3-(3,4-dimethoxyphenyl)-5a-methyl-6-hydroxy-1H-5a,6,7,8,9-pentahydro-1-oxopyrano[4,3-b]benzopyran [3B and 3C]; and cis- and trans-3-(3,4-dimethoxyphenyl)-5a-methyl-6-hydroxy-1H-5a,6,7,8,9-pentahydro-1-oxopyrano[4,3-b]benzopyran [3B and 3C]; 1H-6,7,8,9-tetrahydro-1-oxopyrano[4,3-b]quinoline [24]; 1H-3-methyl-7,8,9, 10-tetrahydropyrano[4,3-c]isoquinolin-1-one [26]; (5aS*, 9aR*, 10R*)-9a, 10-Dihydroxy-3-(3-pyridyl)-1H-5a,6,7,8,9,9a, 10-heptahydro-1-oxopyrano [4,3-b][1] benzopyran [38B]; (5aS, 7S)-7-[2-(1-Pentanoyloxypropyl)]-10-hydroxy-3-(3,4-dimethoxyphenyl)-1H-5a,6,7,8,9,9a, 10-heptahydro-1-oxopyrano [4,3-b][1] benzopyran [37]; (5aS, 7S)-7-Isopropenyl-3-(3,4-dimethoxyphenyl)-1H-5a,6,7,8,9-pentahydro-1-oxopyrano [4,3-b][1] benzopyran [30]; (5aS, 7S)-7-Isopropenyl-3-(3-pyridyl)-1H-5a,6,7,8,9-pentahydro-1-oxopyrano [4,3-b][1] benzopyran [29]; (5aS, 7S)-7-Isopropenyl-3-methyl-1H-5a,6,7,8,9-pentahydro-1-oxopyrano [4,3-b][1] benzopyran [28]; and 3-(Carboxymethyl)-1H-5a,6,7,8,9-pentahydro-1-oxopyrano [4,3-b][1] benzopyran [32].
A most preferred class of compounds of this invention includes compounds selected from the group consisting of 3-(3-pyridyl)-1H-5a,6,7,8,9-pentahydro-1-oxopyrano[4,3-b]benzopyran [2A]; 3-(3,4-dimethoxyphenyl)-1H-5a,6,7,8,9-pentahydro- l -oxopyrano[4,3-b]benzopyran [3A]; cis- and trans-3 -(3,4-dimethoxyphenyl)-5a-methyl-6-formyloxy-1H-5a,6,7,8,9-pentahydro-1-oxopyrano[4,3-b]benzopyran [3D and 3E]; and cis- and trans-3-(3,4-dimethoxyphenyl)-5a-methyl-6-hydroxy-1H-5a,6,7,8,9-pentahydro-1-oxopyrano[4,3-b]benzopyran [3B and 3C]; and 1,8-Di {3-[1H-5a,6,7,8,9-pentahydro-1-oxopyrano [4,3-b] [1] benzopyranyl]}-2,7-octandione [33].
This invention also provides methods as illustrated in Schemes 1, 2, 6, 7, 8 and 9 below for making the above compounds via condensation reactions between an aldehyde of a cyclohexene having R2 and R3 substituents as defined above, and an ortho-oxy-substituted heterocyclic ring having as a para-substituent a reactive group capable of reacting with the xcex2 carbon of the enal function (carbon containing R2) to form the tricyclic product. These anticancer drugs are easy to prepare in large quantities using few steps.
The method comprises contacting:
(a) a compound of the formula: 
wherein X is as defined for Formula I;
wherein R1 is defined as R3 as set forth in Formula I above; and
Z is a reactive group comprising Y (as defined in Formula I above, i.e. O, S or N);
with
(b) a compound having an aldehyde substituent of the formula: 
xe2x80x83wherein:
R2 and R3 are as defined above for Formula I, R6 is defined as R3 for Formula I above, and R4 and R5 are as defined above for Formula I; and T and Z are independently CH, N, S or O
under reaction conditions whereby a condensation reaction takes place between said compounds of paragraphs (a) and (b) whereby reactive groups R3 and Z react with said substituted ene aldehyde to form a compound as defined in the Formula I above.
Compounds of Formula I and Formula 1 where Xxe2x89xa0Y may be made by means known to the art by methods analogous to those disclosed herein. Further, compounds of Formula I and Formula 1 where Txe2x89xa0CH, Zxe2x89xa0CH, R4xe2x89xa0H, or R5xe2x89xa0H may be made by means known to the art by methods analogous to those disclosed herein.
More preferably, the method comprises making a compound of Formula 1 comprising contacting: 
wherein R2 and R3 are as defined for Formula 1 above, with
(b) a compound of the formula: 
xe2x80x83wherein:
R1 is defined as R3 as set forth for Formula 1 above.
Methods are also provided for making compounds of Formula IV above comprising reacting (a) compounds of the formula: 
wherein R1 is defined as R3 as set forth above for Formula I;
with
(b) compounds of the formula: 
wherein R2 and R3 are as defined above for Formula I.
Methods are provided for making compounds of Formula VI above comprising reacting (a) compounds of the formula: 
wherein:
R17 and R18 are independently defined as R3 as set forth for Formula I above;
R19 is CH2R, wherein R is as defined as R3 as set forth for Formula I above;
with
(b) compounds of the formula: 
wherein R1 is defined as R3 as set forth for Formula I above.
Methods are also provided for making a compound of Formula E above comprising reacting: 
wherein R1 is defined as R3 as set forth for Formula I above;
with
(b) a compound of the formula: 
wherein X is I, Br, or Cl, and Ms is methanesulfonyl.
A method is also provided for inhibiting an enzyme selected from the group consisting of acetylcholinesterase and cholesterol acyltransferase in a patient comprising administering to the patient an effective amount of a compound of this invention. An effective amount is an amount capable of effecting measurable inhibition, preferably an amount capable of effecting inhibition equivalent or greater than that of known AChE inhibitor Tacrine or known ACAT inhibitor CP-113,818 (see Examples hereof). As is known to the art, dosage can be adjusted depending on the bioactivity of the particular compound chosen. The compound may be administered in combination with a suitable pharmaceutical carrier such as DMSO, ethyl alcohol, or other carriers known to the art.
Patients include humans, large mammals, livestock animals, pets, and laboratory animals.
A method is also provided for inhibiting macromolecule (e.g., DNA, RNA and protein) synthesis and growth of cancer cells in a patient comprising administering to the patient an effective amount of a compound of this invention. Suitable pharmaceutical carriers may be used for administration of the compound. An effective amount to inhibit macromolecule synthesis or cell growth is an amount sufficient to inhibit macromolecule production or cell growth at least as well as 20(S)-camptothecin (CPT) as measured in standard assays as described in the Examples hereof.
A method is also provided for inhibiting tubulin polymerization in a patient comprising administering to the patient an effective amount of a compound of this invention. Suitable pharmaceutical carriers may be used for administration of the compound. An effective amount is an amount capable of effecting measurable inhibition, preferably an amount capable of effecting inhibition equivalent to known tubulin polymerization inhibitor colchicine.
Methods are also provided herein for prevention of tubulin polymerization, tumor development, inhibiting the rate of tumor growth, and inducing regression of pre-existing tumors comprising administering to a patient an effective amount of a compound of this invention. An effective dosage for each purpose may be readily calculated by those of skill in the art based on effective dosages for inhibition of macromolecule synthesis, optimized and adjusted as required for individual patients.
Interestingly, Tau, which is a major component of the abnormal intracellular tangles of filaments found in the brain of Alzheimer patients, is a non-energy transducing microtubule-associated protein. If tricyclic pyrones bind to tubulin and disrupt microtubule dynamics, they should also decrease or prevent the interactions of Tau and other microtubule-associated proteins with microtubules that are involved in Alzheimer""s disease.
The mechanism of action by which the compounds inhibit cancer cells is unknown; however, a possible mechanism is that the compounds bind selectively and strongly with one of the oxidative enzymes which undergoes oxidation at the C3-C4 double bond to form the corresponding C3-C4 epoxide and this epoxide then subsequently undergoes a ring opening reaction with a nucleophile of DNA, RNA, or enzymes in the cancer cell.